探討PKA亞型抑制核內有絲分裂之機制

曾馨柔

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探討PKA亞型抑制核內有絲分裂之機制

The mechanism of endomitosis inhibited by PKA isoforms

曾馨柔 , Masters　　Advisor：簡偉明　　

繁體中文

核內有絲分裂；細胞週期；環磷酸腺苷；蛋白激酶A ； endomitosis ； cell cycle ； cAMP ； PKA

Abstract │ Reference (97) │ Altmetrics

Abstract 〈TOP〉

多倍化，是指一個細胞擁有超過兩套以上的染色體，這個現象在人體的巨核細胞、肝細胞及肌肉細胞中皆可發現。多倍化現象可藉由核內有絲分裂機制形成，包含細胞分裂的阻斷及染色體的再複製，而巨核細胞便是仰賴這個機制協助產生大量的血小板。當核內有絲分裂產生異常，會導致血小板數量或功能異常。過去研究指出，巨核細胞分化過程中，cyclin B1及CDK1被發現與正常細胞分裂的消失有關，而cyclin D與cyclin E會促使細胞再次進入染色體複製期，提高染色體套數。最近研究更發現環磷酸腺苷-蛋白激酶A(cAMP-PKA)這條訊息路徑會對核內有絲分裂產生抑制作用，然而目前對於cAMP如何抑制核內有絲分裂及影響不同細胞週期階段中的調控分子仍不清楚。因此，本研究利用HEL細胞株作為實驗模型，利用流式細胞儀(Acurri C6)進行觀察，欲了解提高細胞內cAMP濃度會如何影響PMA所誘發的核內有絲分裂及相關調控分子的變化。實驗結果顯示利用腺苷酸環化酶活化劑forskolin提高細胞cAMP濃度確實可以抑制PMA誘發的核內有絲分裂，包含細胞體積及染色體套數下降。接著也發現PMA可以誘發p21表現量增加，且提高cAMP可抑制p21表現量的增加。由於p21可影響細胞週期分子的變化，因此先觀察細胞分裂相關的因子是否受到影響，結果發現處於G2/M期且表現cyclin B1的細胞數有明顯的下降，但cAMP的提高並不影響PMA誘發的效果。進一步觀察細胞中cyclin B1表現量的變化，發現cAMP會抑制該分子晚期表現量的增加。此外，在PMA處理後，cdc2Y15的磷酸化程度會逐漸下降，但cAMP的提高並不會影響PMA誘發的變化。另外，在促進細胞進入染色體複製期分子的部分，發現cyclin D3的表現量在PMA誘發的早期有顯著的增加，而cAMP提高可抑制該分子早期表現量的增加。另外，cyclin E1的表現量在PMA投與後確實會提高，但該提高的表現量並不被cAMP所抑制。故由以上結果推論p21及cyclin D3為主要參與在cAMP造成核內有絲分裂抑制作用的分子。過去研究發現PKA亞型在細胞中的功能不同，因此利用shRNA的方式抑制特定PKA亞型的基因表現，進一步確認cAMP下游的作用分子。結果顯示當完全抑制PKAIIA的表現時，會阻斷cAMP的抑制效果，而部分抑制PKAIIA的表現時，則仍可觀察到cAMP的抑制效果，而抑制PKAIA的表現並不影響cAMP的抑制效果，故可知cAMP是藉由PKAIIA抑制PMA誘發的核內有絲分裂。
核內有絲分裂會影響血小板的生成，若了解cAMP抑制核內有絲分裂的作用機制及下游訊息傳遞路徑的作用分子，對於未來藥物設計的方向及作用奠定基礎，以便運用於血小板疾病及相關疾病的治療。

Parallel Abstract 〈TOP〉

Ployploidy, a cell containing more than two paired set of chromosome, can be observed in megakaryocytes, hepatocytes and muscular cells of human. The phenomenon of polyploidy, also known as endomitosis, is composed of interruption of cytokinesis and re-synthesis of DNA. The abnormality of endomitosis in megakarypoiesis can cause the platelet disorder. In megakarypoiesis, it is reported that cyclin B1 and CDK1 are correlated to interruption of cytokinesis. Cyclin D and cyclin E promote the entrance of S phase and DNA re-synthesis. It’s recently found that cAMP-PKA signaling can inhibit endomitosis through E2A-p21 axis. However, the alternation of those factors at different cell cycle stage in endomitotic cells and the specific mechanism of cAMP inhibition are not completely known. In this study, we observed alternation of cyclins and related factors in PMA-induced endomitosis with and without forskolin-mediated inhibition in HEL cells through Acurri C6 flow cytometry. The results showed that elevation of cAMP levels by forskolin indeed inhibits PMA-induced endomitosis, including the decrease of cell size and cell ploidy. In cytokinesis-related factors, we found number of cyclin B1-expressed-cells at G2/M phase has similar change under PMA treatment and PMA/forskolin co-treatment. However, cAMP can inhibit PMA-induced cyclin B1 expression in these cells at late stage. Furthermore, cdc2Y15 levels have no significantly difference between two groups. In factors related to S-phase entry, cyclin D3 levels are incresed after PMA treatment, but the increase can be inhibited by pre-treatment of cAMP. Moreover, cyclin E is significantly increased after PMA treatment, but forskolin had no effect on the expression induced by PMA. The results showed that cyclin D3 and p21 may be the determinant factors in forskolin-mediated inhibition. Recent studies revealed that cAMP inhibited endomitosis through PKA. To uncover the downstream PKA isoforms of cAMP, we reduced the expression of PKA isoforms with shRNA. The results showed that complete knockdown of PKAIIA can abolish the inhibition on PMA-induced endomitosis by forskolin. However, partial knockdown of PKAIIA can not reverse cAMP-mediated inhibition. Knockdown of PKAIA is not able to withstand the inhibitory effect of cAMP. Therefore, PKAIIA is the downstream factor of cAMP during the inhibition.
Endomitosis is related to the production of functional platelet in human. Knowing the specific mechanisms and the downstream factors may contribute to the treatment strategy and drug design in the future.

Reference ( 97 ) 〈TOP〉

Afdhal N, McHutchison J, Brown R, Jacobson I, Manns M, Poordad F, et al. Thrombocytopenia associated with chronic liver disease. Journal of Hepatology 48(6):1000-7 (2008)
連結：
Aleem E, Kiyokawa H, Kaldis P. Cdc2-cyclin E complexes regulate the G1/S phase transition. Nature cell biology 7(8):831-6 (2005)
連結：
Ando K, Ajchenbaum-Cymbalista F, Griffin JD. Regulation of G1/S transition by cyclins D2 and D3 in hematopoietic cells. PNAS 90(20):9571-5 (1993)
連結：
Baccini V, Roy L, Vitrat N, Chagraoui H, Sabri S, Le Couedic JP, et al. Role of p21(Cip1/Waf1) in cell-cycle exit of endomitotic megakaryocytes. Blood 98(12):3274-82 (2001)
連結：
Bartkova J, Lukas J, Strauss M, Bartek J. Cyclin D3: requirement for G1/S transition and high abundance in quiescent tissues suggest a dual role in proliferation and differentiation. Oncogene 17(8):1027-37 (1998)
連結：

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